Purpose: To determine if the recombinant Asp f 3-based vaccine protects through B or T cell-mediated responses and to elucidate the responsible epitopes. Methods: Animal model groups of CF-1 mice were immunized with rAsp f 3 (residues 15-168, TiterMax as adjuvant), and anti-Asp f 3 titers and isotypes were determined by ELISA and Western blotting. Six weeks post immunization, the animals were immunosuppressed by daily injections of cortisone acetate for 10 days and challenged i.n. with three million viable Aspergillus fumigatus conidia and observed for one week. In separate experiments, T cells were depleted 3 days before challenge using rat GK1.5 antibody. Non-specific rat antibody served as control. Epitope mapping Peptides of trypsin-digested rAsp f 3 were purified by HPLC and analyzed by mass spectrometry. Identified peptides, together with synthetic 20-mer peptides, that cover the Asp f 3 sequence with overlapping ends of five amino acids, were separately co-cultured with splenocytes from immunized survivors. T cell proliferation was determined after magnetic bead isolation and luminescent ATP cell titer quantification. Cytokine production of proliferating T cells was measured by real time PCR, ELISA, intracellular cytokine staining by FACS analysis. Results: Post immunization titers of total IgG (and IgG 2a) against rAsp f 3 did not correlate with survival. However, the survival of immunized mice was significantly higher in groups with no T cell depletion, than in groups with T cell depletion (p = 0.019, n = 10/group). The HPLC-purified tryptic digest peptides PGAFTPVCSAR and HVPEYIEKLPEIR of Asp f 3 had reactivity with total IgG and with IgG 2a from rAsp f 3-immunized survivors in the ELISA. Post immunization IgG from non-surviving mice showed no such reactivity. The digest peptide, HVPEYIEKLPEIR, as well as the synthetic peptide VCSARHVPEYIEKLPEIRAK induced proliferation and IFN-gamma production in T cells that were isolated from surviving animals. Conclusions: Asp f 3-derived protection against aspergillosis in corticosteroid immunosuppressed mice is likely achieved through a T cell-based mechanism. Cytokine induction in cells from immunized survivors supports a Th1 type response. A minimal T cell epitope, identified as HVPEYIEKLPEIR, was discovered. This epitope has the potential for being developed into a subunit vaccine or for use in assays that survey the status of protective immunization.
Full conference title:
4th Advances Against Aspergillosis
- AAA 4th (2010)